METHODS AND COMPOSITIONS FOR USE IN PREPARRING siRNAs

ABSTRACT

Methods and compositions for producing siRNAs, e.g., in the form of a d-siRNA composition, from dsRNAs are provided. In the subject methods, a dsRNA is contacted with a composition that includes an activity that cleaves dsRNA into siRNAs, where the composition efficiently cleaves dsRNA into siRNAs. siRNAs produced by the subject methods find use in a variety of applications, particularly in applications where the specific reduction or silencing of a gene is desired. Also provided are kits for use in practicing the subject invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/427,758 filed Apr. 30, 2003 which claims priority (pursuant to 35U.S.C. § 119 (e)) to the filing date of the U.S. Provisional PatentApplication Ser. No. 60/400,655 filed Aug. 1, 2002 and U.S. ProvisionalPatent Application Ser. No. 60/377,704 filed May 3, 2002; thedisclosures of which are herein incorporated by reference.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contract GM46383awarded by the National Institutes of Health. The Government has certainrights in this invention.

INTRODUCTION

1. Field of the Invention

The field of this invention is genomics.

2. Background of the Invention

RNAi has become the method of choice for loss-of-function investigationsin numerous systems including, C. elegans, Drosophila, fungi, plants,and even mammalian cell lines. To specifically silence a gene in mostmammalian cell lines, small interfering RNAs (siRNA) are used becauselarge dsRNAs (>30 bp) trigger the interferon response and causenonspecific gene silencing. Currently, siRNAs are produced by chemicalsynthesis, by in vitro transcription from a short DNA template, or bytransfection of DNA plasmids that give rise to hairpin RNAs in vivo.

All of these approaches are capable of gene silencing, but not withoutproblems. First, the chemical synthesis of siRNAs is very expensive. Thecurrent price of a single custom-synthesized siRNA is several hundreddollars, and three to eight siRNAs may be required to attain a highprobability of silencing any particular gene. In addition, the rules forwhat makes an effective siRNA are not well worked out, and consequentlythere is a substantial chance that any single 21 nucleotide regionselected from the mRNA will be ineffective in initiating mRNA cleavageor inhibiting translation. In vitro transcribed-siRNAs andplasmid-encoded hairpin-siRNAs are less expensive, but like chemicalsynthesis there is a chance that the 21-nucleotide target will beineffective for gene silencing. Moreover, such methods do not scale upeasily for screens, since for each member of the library, one or moreoligonucleotides would need to be individually designed and synthesized.In addition, single siRNAs cause cleavage of the target mRNA at a singlesite, opening the possibility that the remaining 3′-fragment will betranslated. The resulting N-terminal truncated protein may act as adominant negative or constitutively active protein rather than as a trueprotein-null. An inexpensive, efficient method for generating a largenumber of different siRNAs from any given mRNA or from a mix of mRNAswould obviate these problems.

In order to improve the efficacy of RNAi use in mammalian cell lines, aninexpensive and efficient method of generating a large number ofdifferent siRNAs is needed. The present invention satisfies this need.

Relevant Literature

Published PCT applications of interest include WO 01/68836.

SUMMARY OF THE INVENTION

Methods and compositions for producing siRNAs, e.g., in the form of ad-siRNA composition, from dsRNAs are provided. In the subject methods, adsRNA is contacted with a composition that includes an activity thatcleaves dsRNA into siRNAs, where the composition efficiently cleavesdsRNA into siRNAs. siRNAs produced by the subject methods find use in avariety of applications, particularly in applications where the specificreduction or silencing of a gene is desired. Also provided are kits foruse in practicing the subject invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A to 1C. r-Dicer activity in vitro. FIG. 1A: Analysis of r-Dicerexpression and in vitro activity. Lysates from uninfected andDicer-infected Hi5 cells were fractionated on a cobalt-Sepharoseaffinity resin. The upper panels are from a Coomassie-stained gelshowing the 225 kDa Dicer protein and other proteins present in thevarious purification fractions. The middle panels are from an immunoblotwith an anti-T7-epitope antibody that recognizes the tag at theN-terminus of r-Dicer. The bottom panels are from an ethidium-stainednative polyacrylamide gel showing the 500 bp dsRNA Dicer substrate andthe 22 bp d-siRNAs produced by active r-Dicer. FIG. 1B: Copurificationof r-Dicer protein and activity on Q-Sepharose. Affinity-purifiedr-Dicer was further purified on Q-Sepharose and assayed for r-Dicerprotein (upper panel) and activity (bottom panel). FIG. 1C: Cofactorrequirements and substrate specificity of r-Dicer. r-Dicer was incubatedwith under standard reaction conditions with 500 bp dsRNA, a 500 bpssRNA, or a 600 bp dsDNA (lanes 1-3). r-Dicer was also incubated with500 bp dsRNA with various additions to (RNasin) or omissions from (ATPand or Mg²⁺) the reaction mix (lanes 4-7).

FIGS. 2A to H. Production of pools of siRNAs by purified recombinantDicer. (A) The size of d-siRNAs produced by r-Dicer. Three different˜500 bp dsRNAs were incubated with r-Dicer. The reactions were thenelectrophoresed on an 18% sequencing gel. The radiolabeled siRNA andd-siRNAs were visualized by exposure to a phosphorimager and the size ofthe d-siRNAs were determined by comparison to a synthetic 21 bp siRNAand to a single base pair ladder. (B) Phosphorylation status of the5′-end of various d-siRNAs. A synthetic siRNA or various d-siRNAs wereincubated in shrimp alkaline phosphatase (SAP) buffer either in thepresence or absence of SAP, followed by T4 polynucleotide kinase and[γ-³²P]ATP. Reactions were electrophoresed on a 15% nativepolyacrylamide gel. The amount of radioactivity present was visualizedusing a phosphorimager (upper panel) and the presence of the siRNA ord-siRNA was confirmed by staining with ethidium bromide (lower panel).(C-H) Analysis of the efficiency with which r-Dicer cleaves the ˜500 bpdsRNA substrate into 20-21 bp d-siRNA products. Increasing amounts ofr-Dicer were incubated with a constant amount (1 μg) of the ˜500 bpsubstrate. The reactions were stopped at 5, 10 and 24 hours. Substrateand products were separated on a 15% native polyacrylamide gel and thenvisualized (C) and quantified (D-H) with a Phosphorimager.

FIGS. 3A to H. d-siRNAs specifically silence luciferase expression inHEK 293 cells. (A) Quantity, quality and purity of siRNAs and d-siRNAs.Various ˜500 bp dsRNAs were cleaved by r-Dicer to generate d-siRNAs. Thecontaminating reaction components and ˜500 bp dsRNA were separated fromthe d-siRNAs by a series of spin columns. Chemically synthesized siRNAs(lanes 1 and 2), purified d-siRNAs (lanes 4, 6, and 8) and a portion ofthe unpurified dicing reaction (lanes 3, 5, and 7) were electrophoresedon a 15% native polyacrylamide gel. RNAs were visualized by stainingwith ethidium bromide. (B, C) Absolute firefly and Renilla luciferaseactivity. Firefly (Photinus pyralis, Pp-luc, GL3) and sea pansy (Renillareinformis, Rr-luc, RL) luciferase expression vectors were cotransfectedwith various siRNAs or d-siRNAs (30 nmol per L medium). The firefly (B)and Renilla (C) luciferase activities were determined individually. (D)Normalized luciferase activity. Either the Pp-luc/Rr-luc (grey bars) orthe Rr-luc/Pp-luc ratio (white bars), depending on which luciferase wasthe target, was calculated. The plotted data was averaged from threeindependent experiments±S.E. (E-G) Absolute (E, F) and normalized (G)firefly and Renilla luciferase activity. The firefly (Photinus pyralis,Pp-luc, GL3) and sea pansy (Renilla reinformis, Rr-luc, RL) luciferaseexpression vectors were cotransfected with either various concentrationsof GL3 or GFP siRNAs or d-siRNAs. Concentrations were taken as nmol perL of medium. The firefly (E) and Renilla (F) luciferase activities weredetermined individually (dark gray bars indicate cells transfected withsiRNAs or d-siRNAs targeting GL3; light gray bars indicate cellstransfected with an irrelevant GFP siRNA or d-siRNA). The Pp-luc/Rr-lucratio (G) was calculated for cells transfected with GL3 siRNAs ord-siRNAs (dark gray bars) and for cells transfected with irrelevant GFPsiRNAs or d-siRNAs (light gray bars). The plotted data was averaged fromthree independent experiments±S.E. (H) elF2α phosphorylation in cellstreated with synthetic and diced siRNAs. The various cellular lysateswere subjected to immunoblotting for elF2α phosphorylation (top) or, asa loading control, actin (bottom). “Positive control” denotes cellstreated with 15 nM 500 bp dsRNA; “negative control” denotes untreatedcells.

FIGS. 4A to B. d-siRNAs can silence endogenous genes. (A) Cyclin E1 issilenced in a dose dependent fashion. HEK 293 cells were transfectedwith two different pools of d-siRNAs, one complementary to cyclin E1 andanother to Renilla luciferase. After 72 h, cells were lysed andsubjected to immunoblotting with various antibodies. (B) Cdc25C d-siRNAssilence Cdc25C expression without affecting Cdc25A expression. HEK 293cells were transfected for 72 h with two different pools of d-siRNAs,one complementary to Cdc25C and another to B-Raf. Immunoblotting showsamounts of the Cdc25C, Cdc25A, actin (loading control), andphospho-elF2α.

DEFINITIONS

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a genomic integrated vector, or“integrated vector”, which can become integrated into the chromsomal DNAof the host cell. Another type of vector is an episomal vector, i.e., anucleic acid capable of extra-chromosomal replication. Vectors capableof directing the expression of genes to which they are operativelylinked are referred to herein as “expression vectors”. In the presentspecification, “plasmid” and “vector” are used interchangeably unlessotherwise clear from the context.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as applicable tothe embodiment being described, single-stranded (such as sense orantisense) and double-stranded polynucleotides.

As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding a polypeptide ofthe present invention, including both exon and (optionally) intronsequences. A “recombinant gene” refers to nucleic acid encoding suchregulatory polypeptides, that may optionally include intron sequencesthat are derived from chromosomal DNA. The term “intron” refers to a DNAsequence present in a given gene that is not translated into protein andis generally found between exons. As used herein, the term“transfection” means the introduction of a nucleic acid, e.g., anexpression vector, into a recipient cell by nucleic acid-mediated genetransfer.

A “protein coding sequence” or a sequence that “encodes” a particularpolypeptide or peptide, is a nucleic acid sequence that is transcribed(in the case of DNA) and is translated (in the case of mRNA) into apolypeptide in vitro or in vivo when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, cDNA from procaryotic or eukaryoticmRNA, genomic DNA sequences from procaryotic or eukaryotic DNA, and evensynthetic DNA sequences. A transcription termination sequence willusually be located 3′ to the coding sequence.

Likewise, “encodes”, unless evident from its context, will be meant toinclude DNA sequences that encode a polypeptide, as the term istypically used, as well as DNA sequences that are transcribed intoinhibitory antisense molecules.

The term “loss-of-function”, as it refers to genes inhibited by thesubject RNAi method, refers a diminishment in the level of expression ofa gene when compared to the level in the absense of dsRNA constructs.

The term “expression” with respect to a gene sequence refers totranscription of the gene and, as appropriate, translation of theresulting mRNA transcript to a protein. Thus, as will be clear from thecontext, expression of a protein coding sequence results fromtranscription and translation of the coding sequence.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

As used herein, the terms “transduction” and “transfection” are artrecognized and mean the introduction of a nucleic acid, e.g., anexpression vector, into a recipient cell by nucleic acid-mediated genetransfer. “Transformation”, as used herein, refers to a process in whicha cell's genotype is changed as a result of the cellular uptake ofexogenous DNA or RNA, and, for example, the transformed cell expresses adsRNA contruct.

“Transient transfection” refers to cases where exogenous DNA does notintegrate into the genome of a transfected cell, e.g., where episomalDNA is transcribed into mRNA and translated into protein.

A cell has been “stably transfected” with a nucleic acid construct whenthe nucleic acid construct is capable of being inherited by daughtercells.

As used herein, a “reporter gene construct” is a nucleic acid thatincludes a “reporter gene” operatively linked to at least onetranscriptional regulatory sequence. Transcription of the reporter geneis controlled by these sequences to which they are linked. The activityof at least one or more of these control sequences can be directly orindirectly regulated by the target receptor protein. Exemplarytranscriptional control sequences are promoter sequences. A reportergene is meant to include a promoter-reporter gene construct that isheterologously expressed in a cell.

As used herein, “transformed cells” refers to cells that havespontaneously converted to a state of unrestrained growth, i.e., theyhave acquired the ability to grow through an indefinite number ofdivisions in culture. Transformed cells may be characterized by suchterms as neoplastic, anaplastic and/or hyperplastic, with respect totheir loss of growth control. For purposes of this invention, the terms“transformed phenotype of malignant mammalian cells” and “transformedphenotype” are intended to encompass, but not be limited to, any of thefollowing phenotypic traits associated with cellular transformation ofmammalian cells: immortalization, morphological or growthtransformation, and tumorigenicity, as detected by prolonged growth incell culture, growth in semi-solid media, or tumorigenic growth inimmuno-incompetent or syngeneic animals.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

As used herein, “immortalized cells” refers to cells that have beenaltered via chemical, genetic, and/or recombinant means such that thecells have the ability to grow through an indefinite number of divisionsin culture.

The “growth state” of a cell refers to the rate of proliferation of thecell and the state of differentiation of the cell.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and compositions for producing siRNAs, e.g., in the form of ad-siRNA composition, from dsRNAs are provided. In the subject methods, adsRNA is contacted with a composition that includes an activity thatcleaves dsRNA into siRNAs, where the composition efficiently cleavesdsRNA into siRNAs. siRNAs produced by the subject methods find use in avariety of applications, particularly in applications where the specificreduction or silencing of a gene is desired. Also provided are kits foruse in practicing the subject invention.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, representativemethods, devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing the components that aredescribed in the publications which might be used in connection with thepresently described invention.

In further describing the subject invention, the subject methods aredescribed first in greater detail, followed by a description of theproduct d-siRNA compositions produced thereby, a review of variousrepresentative applications, including therapeutic applications, inwhich the subject invention finds use. Finally, kits that find use inpracticing the subject invention are discussed.

Methods

As summarized above, the subject invention provides methods ofefficiently producing siRNA from dsRNA. More specifically, the subjectinvention provides methods of producing a plurality of siRNA moleculesfrom a parent dsRNA molecule. By plurality is meant at least 2, usuallyat least about 5, and more usually at least about 10, where the numberof distinct siRNA molecules produced from a given parent dsRNA moleculein the subject methods will often depend on the length of the parentdsRNA molecule, but may be as high as about 25 or higher, e.g., about100, about 400 or higher. The siRNA product molecules in manyembodiments range in length from about 10 to about 30-35 residues, e.g.,from about 15 to about 25 residues, including from about 20 to 23residues, where molecules of 12, 15, 18, 20, 21, 22, 25 and 29 residuesin length are of particular interest in certain embodiments.

The length of the parent dsRNA molecules that are employed in thesubject methods may vary, but generally the length is at least about 300bp, usually at least about 500 bp and more usually at least about 1000bp, where the length may be as long as about 2000 bp or longer, butoften does not exceed about 8000 bp, e.g., 6000 bp.

The dsRNA construct may comprise two hybridized strands of polymerizedribonucleotide. It may include modifications to either thephosphate-sugar backbone or the nucleoside. For example, thephosphodiester linkages of natural RNA may be modified to include atleast one of a nitrogen or sulfur heteroatom. Modifications in RNAstructure may be tailored to allow specific genetic inhibition whileavoiding a general panic response in some organisms which is generatedby dsRNA. Likewise, bases may be modified to block the activity ofadenosine deaminase. The dsRNA construct may be produced enzymaticallyor by partial/total organic synthesis, any modified ribonucleotide canbe introduced by in vitro enzymatic or organic synthesis.

The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. dsRNAconstructs containing a nucleotide sequence identical to a portion of atarget gene may be employed for inhibition. RNA sequences withinsertions, deletions, and single point mutations relative to the targetsequence are also of interest for inhibition applications. Thus,sequence identity may be optimized by sequence comparison and alignmentalgorithms known in the art (see Gribskov and Devereux, SequenceAnalysis Primer, Stockton Press, 1991, and references cited therein) andcalculating the percent difference between the nucleotide sequences by,for example, the Smith-Waterman algorithm as implemented in the BESTFITsoftware program using default parameters (e.g., University of WisconsinGenetic Computing Group). Greater than 90% sequence identity, or even100% sequence identity, between the inhibitory RNA and the portion ofthe target gene may be of interest. Alternatively, the duplex region ofthe RNA may be defined functionally as a nucleotide sequence that iscapable of hybridizing with a portion of the target gene transcriptunder stringent conditions (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed bywashing; or conditions that are at least as stringent as theserepresentative conditions). The length of the identical nucleotidesequences may be, for example, at least about 25, about 50, about 100,about 200, about 300 or about 400 bases or longer. In certainembodiments, the dsRNA construct is from about 400 to about 800 bases inlength. In certain embodiments 100% sequence identity between the RNAand the target gene is not required to practice inhibition applicationsof the invention. Thus the invention has the advantage of being able totolerate sequence variations that might be expected due to geneticmutation, strain polymorphism, or evolutionary divergence.

The dsRNA construct employed as the parent dsRNA in the presentapplications may be synthesized either in vivo or in vitro. Endogenouspolymerase of the cell may mediate transcription in vivo, or cloned RNApolymerase can be used for transcription in vivo or in vitro. Fortranscription from a transgene in vivo or an expression construct, aregulatory region (e.g., promoter, enhancer, silencer, splice donor andacceptor, polyadenylation) may be used to transcribe the dsRNA strand(or strands). Inhibition may be targeted by specific transcription in anorgan, tissue, or cell type; stimulation of an environmental condition(e.g., infection, stress, temperature, chemical inducers); and/orengineering transcription at a developmental stage or age. The RNAstrands may or may not be polyadenylated; the RNA strands may or may notbe capable of being translated into a polypeptide by a cell'stranslational apparatus. The dsRNA construct may be chemically orenzymatically synthesized by manual or automated reactions. The dsRNAconstruct may be synthesized by a cellular RNA polymerase or abacteriophage RNA polymerase (e.g., T3, T7, SP6). The use and productionof an expression construct are known in the art (see WO 97/32016; U.S.Pat. Nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693; andthe references cited therein). If synthesized chemically or by in vitroenzymatic synthesis, the RNA may be purified prior to introduction intothe cell. For example, RNA can be purified from a mixture by extractionwith a solvent or resin, precipitation, electrophoresis, chromatographyor a combination thereof. Alternatively, the dsRNA construct may be usedwith no or a minimum of purification to avoid losses due to sampleprocessing. The dsRNA construct may be dried for storage or dissolved inan aqueous solution. The solution may contain buffers or salts topromote annealing, and/or stabilization of the duplex strands.

In practicing the subject methods, the parent dsRNA molecule asdescribed above, is contacted with a composition enriched for a proteinhaving an activity that cleaves the parent dsRNA substrate intofragments having siRNA activity, i.e., into the desired siRNApopulation. In other words, the first step of the subject methods is toproduce a reaction composition by combining the parent dsRNA and acomposition enriched for a protein having an activity that cleaves theparent dsRNA substrate into fragments having siRNA activity.

The dsRNA cleaving protein enriched composition employed in the subjectmethods is a composition that contains a sufficient amount of an enzymethat cleaves dsRNA into siRNA. In many embodiments, the dsRNA cleavingenzyme present in the composition is a Dicer protein. Dicer is a memberof the RNAse III family of nucleases that specifically cleave dsRNA andis evolutionarily conserved in worms, flies, plants, fungi and mammals.The enzyme has a distinctive structure that includes a helicase domainand dual RNAse III motifs. Dicer also contains a region of homology tothe RDE1/QDE2/ARGONAUTE family of proteins, which have been geneticallylinked to RNAi in lower eukaryotes.

Specific Dicer proteins of interest include, but are not limited to:

A) a protein which cleaves dsRNA into siRNA and has an amino acidsequence at least 50 percent identical, and more preferably at least 75,85, 90 or 95 percent identical to the sequence of human dicer ordrosophila dicer (as published in published PCT publication no WO01/68836 and its counterpart published U.S. Application No. 20020162126,the disclosure of the latter of which is herein incorporated byreference (see sequence id nos 2 and 4 of these publications for thehuman and mouse sequences, respectively)); and/or which can be encodedby a nucleic acid which hybridizes under wash conditions of 2×SC at 22°C., and more preferably 0.2×SSC at 65° C., to a nucleic acid (e.g., atleast about 25 nt long, such as at least about 50 nt long, including atleast about 100 nt long) encoding the above human or drosophila Dicerproteins, (as published in published PCT publication no WO 01/68836 andits counterpart published U.S. Application No. 20020162126, thedisclosure of the latter of which is herein incorporated by reference(see sequence id nos 1 and 3 of these publications for the human andmouse sequences, respectively)); the C. elegans dicer (see e.g.,NM_(—)060086) and the like.

Additional specific Dicer proteins and nucleic acids encoding the samewhich are of interest include but are not limited to those havingsequences deposited with Genbank and having the following accessionnos.: NM_(—)177438; NM_(—)030621; XM_(—)216776.

Also of interest is are bacterial RNAse III proteins that exhibit thedesired activity, such as the E.coli RNAseIII protein employed in Yanget al., Proc. Nat'l Acad. Sci. USA (Jul. 23, 2002) 99:9942-99947).

Also of interest in the subject methods is the use of homologs of theabove specific Dicer proteins, e.g., from other animal species, wheresuch homologs or proteins may be from a variety of different types ofspecies, usually mammals, e.g., rodents, such as mice, rats; domesticanimals, e.g. horse, cow, dog, cat; and primates, e.g., monkeys,baboons, humans etc. By homolog is meant a protein having at least about35%, usually at least about 40% and more usually at least about 60%amino acid sequence identity to the specific human transcriptionrepressor factors as identified above, where sequence identity isdetermined using the algorithm described supra.

Also of interest are for use in the subject methods are Dicer proteinsthat are encoded by nucleic acids that are homologous to the aboveprovided nucleic acids, at least with respect to the coding regionsthereof. The source of homologous nucleic acids to those specificallylisted above may be any mammalian species, e.g., primate species,particularly human; rodents, such as rats and mice, canines, felines,bovines, equines, etc; as well as non-mammalian species, e.g., yeast,nematodes, etc. Sequence similarity is calculated based on a referencesequence, which may be a subset of a larger sequence, such as aconserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 nt long, more usually atleast about 30 nt long, and may extend to the complete sequence that isbeing compared. Algorithms for sequence analysis are known in the art,such as BLAST, described in Altschul et al. (1990), J. Mol. Biol.215:403-10 (using default settings, i.e. parameters w=4 and T=17).Unless indicated otherwise, the sequence similarity values reportedherein are those determined using the above referenced BLAST programusing default settings. Of particular interest in certain embodimentsare nucleic acids including a sequence substantially similar to thespecific nucleic acids identified above, where by substantially similaris meant having sequence identity to this sequence of at least about90%, usually at least about 95% and more usually at least about 99%.

Also of interest are nucleic acids that hybridize to the above describednucleic acids under stringent conditions. An example of stringenthybridization conditions is hybridization at 50° C. or higher and0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C. Stringenthybridization conditions are hybridization conditions that are at leastas stringent as the above representative conditions. Other stringenthybridization conditions are known in the art and may also be employedto identify nucleic acids of this particular embodiment of theinvention.

In many embodiments, the composition is one that includes recombinantDicer or an active fragment thereof, i.e., a fragment that retains dsRNAcleaving activity as described above (see e.g., XM_(—)028306 andXM_(—)028307). By recombinant Dicer is meant Dicer that is producedusing recombinant nucleic acid protocols.

Recombinant Dicer may be produced using expression vectors containing anucleic acid encoding a Dicer polypeptide, operably linked to at leastone transcriptional regulatory sequence. Operably linked is intended tomean that the nucleotide sequence is linked to a regulatory sequence ina manner that allows expression of the nucleotide sequence. Regulatorysequences are art-recognized and are selected to direct expression ofthe subject Dicer proteins. Accordingly, the term transcriptionalregulatory sequence includes promoters, enhancers and other expressioncontrol elements. Such regulatory sequences are described in Goeddel;Gene Expression Technology: Methods hi Enzymology 185, Academic Press,San Diego, Calif. (1990). For instance, any of a wide variety ofexpression control sequences, sequences that control the expression of aDNA sequence when operatively linked to it, may be used in these vectorsto express DNA sequences encoding Dicer polypeptides to recombinantlyproduce Dicer. Such useful expression control sequences, include, forexample, a viral LTR, such as the LTR of the Moloney murine leukemiavirus, the early and late promoters of SV40, adenovirus orcytomegalovirus immediate early promoter, the lac system, the trpsystem, the TAG or TRC system, T7 promoter whose expression is directedby T7 RNA polymerase, the major operator and promoter regions of phageX, polyhedron promoter, the control regions for fd coat protein, thepromoter for 3-phosphoglycerate kinase or other glycolytic enzymes, thepromoters of acid phosphatase, e.g., Pho5, the promoters of the yeasta-mating factors, the polyhedron promoter of the baculovirus system andother sequences known to control the expression of genes of prokaryoticor eukaryotic cells or their viruses, and various combinations thereof.It should be understood that the design of the expression vector maydepend on such factors as the choice of the host cell to be transformedand/or the type of protein desired to be expressed.

Moreover, the vector's copy number, the ability to control that copynumber and the expression of any other proteins encoded by the vector,such as antibiotic markers, should also be considered.

The recombinant Dicer genes can be produced by ligating a nucleic acidencoding a Dicer polypeptide into a vector suitable for expression ineither prokaryotic cells, eukaryotic cells, or both. Expression vectorsfor production of recombinant forms of the subject Dicer polypeptidesinclude plasmids and other vectors. For instance, suitable vectors forthe expression of a Dicer polypeptide include plasmids of the types:pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids,pBTac-derived plasmids and pUC-derived plasmids for expression inprokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIPS, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, a Dicer polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of a Dicer gene.

The mammalian expression vectors of certain embodiments contain bothprokaryotic sequences, to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and17.

As such, the subject proteins and polypeptides may be expressed inprokaryotes or eukaryotes in accordance with conventional ways,depending upon the purpose for expression. For large scale production ofthe protein, a unicellular organism, such as E. coli B. subtilis, S.cerevisiae, insect cells in combination with baculovirus vectors, orcells of a higher organism such as vertebrates, particularly mammals,e.g. COS 7 cells, may be used as the expression host cells. In somesituations, it is desirable to express the gene in eukaryotic cells,where the encoded protein will benefit from native folding andpost-translational modifications. Small peptides can also be synthesizedin the laboratory. Polypeptides that are subsets of the completesequence may be used to identify and investigate parts of the proteinimportant for function.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems. Representativesystems from each of these categories is are provided below:

Bacteria. Expression systems in bacteria include those described inChang et al., Nature (1978) 275:615; Goeddel et al., Nature (1979)281:544; Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0 036,776;U.S. Pat. No. 4,551,433; DeBoer et al., Proc. Natl. Acad. Sci. (USA)(1983) 80:21-25; and Siebenlist et al., Cell (1980) 20:269.

Yeast. Expression systems in yeast include those described in Hinnen etal., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al., J.Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142;Kunze et al., J. Basic Microbiol. (1985) 25:141; Gleeson et al., J. Gen.Microbiol. (1986) 132:3459; Roggenkamp et al., Mol. Gen. Genet. (1986)202:302; Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt etal., J. Bacteriol. (1983) 154:737; Van den Berg et al., Bio/Technology(1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141; Cregg etal., Mol. Cell. Biol. (1985) 5:3376; U.S. Pat. Nos. 4,837,148 and4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr.Genet. (1985) 10:380; Gaillardin et al., Curr. Genet. (1985) 10:49;Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284-289;Tilburn et al., Gene (1983) 26:205-221; Yelton et al., Proc. Natl. Acad.Sci. (USA) (1984) 81:1470-1474; Kelly and Hynes, EMBO J. (1985)4:475479; EP 0 244,234; and WO 91/00357.

Insect Cells. Expression of heterologous genes in insects isaccomplished as described in U.S. Pat. No. 4,745,051; Friesen et al.,“The Regulation of Baculovirus Gene Expression”, in: The MolecularBiology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0 127,839; EP 0155,476; and Vlak et al., J. Gen. Virol. (1988) 69:765-776; Miller etal., Ann. Rev. Microbiol. (1988) 42:177; Carbonell et al., Gene (1988)73:409; Maeda et al., Nature (1985) 315:592-594; Lebacq-Verheyden etal., Mol. Cell. Biol. (1988) 8:3129; Smith et al., Proc. Natl. Acad.Sci. (USA) (1985) 82:8844; Miyajima et al., Gene (1987) 58:273; andMartin et al., DNA (1988) 7:99. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts aredescribed in Luckow et al., Bio/Technology (1988) 6:47-55, Miller etal., Generic Engineering (1986) 8:277-279, and Maeda et al., Nature(1985) 315:592-594.

Mammalian Cells. Mammalian expression is accomplished as described inDijkema et al., EMBO J. (1985) 4:761, Gorman et al., Proc. Natl. Acad.Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985) 41:521 and U.S.Pat. No. 4,399,216. Other features of mammalian expression arefacilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:44,Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat. Nos.4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195,and U.S. RE 30,985.

Once the source of the protein is identified and/or prepared, e.g. atransfected host expressing the protein is prepared, the protein is thenpurified to produce the desired repressor protein comprisingcomposition. Any convenient protein purification procedures may beemployed, where suitable protein purification methodologies aredescribed in Guide to Protein Purification, (Deuthser ed.) (AcademicPress, 1990). For example, a lysate may be prepared from the originalsource, e.g. naturally occurring cells or tissues that express thesubject repressor proteins or the expression host expressing the subjectrepressor proteins, and purified using HPLC, exclusion chromatography,gel electrophoresis, affinity chromatography, and the like.

In many embodiments, the Dicer composition employed in the subjectmethods is one that is produced recombinantly from a host cell that doesnot endogenously produce Dicer. As such, the Dicer composition is freeof components that are typically associated with Dicer and found inlysates prepared from cells that endogenously produce Dicer. Forexample, in certain embodiments the Dicer compositions employed in thesubject methods are free of the RISC protein and/or the Argonautprotein, where these proteins are known in the art and described in WO01/68836.

A feature of the subject invention is that the dsRNA cleavingcomposition employed in the subject methods is not a compositionproduced by immunoprecipitation protocols, i.e., it is not an IP Dicerpreparation. As such, the Dicer preparation employed in the subjectmethods is one that is free of those components that are typicallypresent in IP produced preparations. Such components which are notpresent in the Dicer preparations employed in the present inventioninclude: solid phase supports, e.g., beads, as well as other proteinsthat may be coprecipitated, e.g., that are bound by Dicer ornonspecifically precipitated with Dicer.

In the Dicer compositions employed in the subject invention, the amountof Dicer present in the composition may vary, but is typically at leastabout 20 ng/μl, usually at least about 40 ng/μl and more usually atleast about 160 ng/μl . As such, the composition typically has a Diceractivity, as measured by the assay described in the experimentalsection, below, of at least about 10% cleavage of large dsRNA, usuallyat least about 25% cleavage of large dsRNA and more usually at leastabout 75% cleavage of large dsRNA, where the activity may be as high asabout 90% cleavage of dsRNA or higher.

Typically, the composition is an aqueous composition of Dicer, where thecomposition may include one or more additional components, e.g.,buffers, salts like NaCl, MgCl₂, EDTA, DTT, ATP and the like.

In certain embodiments, the composition employed includes a singlenuclease activity, e.g., DICER. In yet other embodiments, thecomposition employed includes two or more different activities, e.g.,dicer, argonaut, etc.

As summarized above, the first step in the subject methods is to contactthe Dicer composition with the substrate dsRNA to produce a reactioncomposition that is then maintained under conditions sufficient toproduce the desired siRNA product. In many embodiments, the subjectmethods are in vitro methods, by which is meant that they occur in acell free environment, e.g., not inside of a cell or in the presence ofcells. As such, the subject methods are typically performed in a testtube or other analogous in vitro environment. More specifically, thereaction composition produced by combining the substrate parent dsRNAand the cleaving protein enriched composition, as described above, isproduced in vitro, i.e., outside of a cell.

The reaction mixture produced by combining the Dicer preparation and thesubstrate dsRNA typically includes a sufficient amount of Mg²⁺ to ensureadequate Dicer activity, where the amount of Mg²⁺ typically ranges fromabout 0.5 mM to about 1.0 mM, usually from about 2.5 mM to about 5.0 mM.A feature of the reaction mixtures or compositions in many embodimentsof the subject invention is that the reaction mixture is free of ATP,and in other embodiments 1 mM ATP is used.

The reaction mixture is typically maintained under incubation conditionssufficient to produce the desired RNAi product. The reaction mixture istypically maintained at a temperature that ranges from about 30 to about37° C., usually from about 35 to about 37° C., for a period of timeranging from about 5 hrs to about 10 hrs, usually from about 16 hrs toabout 24 hrs. Where desired, the mixture may be agitated/stirred duringincubation.

Incubation of the reaction mixture as described above results in theproduction of a siRNA product. The siRNA product (d-siRNA) is aplurality of distinct siRNA molecules that range in length from about 10to about 30-35 residues, e.g., from about 15 to about 25 residues,including from about 20 to 23 residues, where molecules of 12, 15, 18,20, 21, 22, 25 and 29 residues in length are of particular interest incertain embodiments. The number of different or distinct siRNA moleculesproduced is at least about 2, usually at least about 5, and more usuallyat least about 10, where the number of distinct siRNA molecules producedfrom a given parent dsRNA molecule in the subject methods will oftendepend on the length of the parent dsRNA molecule, but may be as high asabout 25 or higher, e.g., about 100, about 400 or higher. In certainembodiments, each distinct siRNA member making up a given d-siRNAproduct composition ranges in length from about 20-21 to 22 nt, and thenumber of distinct siRNA members is at least about 25, where this numbermay be up to and including 100 or more.

The above methods result in the efficient production of a d-siRNAproduct made up of a plurality of distinct siRNA molecules from a parentdsRNA molecule. By efficient is meant that at least the majority of theparent substrate dsRNA is cleaved to product siRNA by the subjectmethods, where the amount of dsRNA that is cleaved is often at leastabout 60 number %, e.g., at least about 70, 75, 80, 90, 95, etc., number%, as determined using any convenient protocol, e.g., by comparing theamount (e.g., as measured by electrophoresis) of parent dsRNA present inthe reaction mixture before and after contact with the Dicerpreparation. The siRNA product, i.e., the d-siRNA composition, producedby the subject methods may be used as is or further processed prior touse, e.g., separated from other components of the reaction mixture,e.g., the Dicer protein, other proteins, remaining large dsRNAs, salts,buffers, NTPsetc. Any convenient separation protocol may be employed,including gel purification, chromatographical separation based onmolecular weight or affinity resins, and classical precipitation and thelike.

The resultant d-siRNA products produced by the methods as describedabove find use in a variety of different applications, whererepresentative applications are review below.

Utility

The d-siRNA product of the subject methods finds use in a variety ofdifferent applications. Representative applications include, but are notlimited to: Drug screening/target validation, large scale functionallibrary screening, silencing single genes, silencing families of genesi.e., ser/thr kinases, phosphatases, membrane receptors etc. and thelike. The subject d-siRNA products also find use in therapeuticapplications, as described in greater detail separately below.

One representative utility of the present invention is as a method ofidentifying gene function in an organism, especially higher eukaryotescomprising the use of the product d-siRNA to inhibit the activity of atarget gene of previously unknown function. Instead of the timeconsuming and laborious isolation of mutants by traditional geneticscreening, functional genomics using the subject product d-siRNAdetermines the function of uncharacterized genes by employing thed-siRNA to reduce the amount and/or alter the timing of target geneactivity. The product d-siRNA can be used in determining potentialtargets for pharmaceutics, understanding normal and pathological eventsassociated with development, determining signaling pathways responsiblefor postnatal development/aging, and the like. The increasing speed ofacquiring nucleotide sequence information from genomic and expressedgene sources, including total sequences for mammalian genomes, can becoupled with use of the product d-siRNA to determine gene function in acell or in a whole organism. The preference of different organisms touse particular codons, searching sequence databases for related geneproducts, correlating the linkage map of genetic traits with thephysical map from which the nucleotide sequences are derived, andartificial intelligence methods may be used to define putative openreading frames from the nucleotide sequences acquired in such sequencingprojects.

A simple representative assay inhibits gene expression according to thepartial sequence available from an expressed sequence tag (EST).Functional alterations in growth, development, metabolism, diseaseresistance, or other biological processes would be indicative of thenormal role of the ESTs gene product.

The ease with which the product d-siRNA construct can be introduced intoan intact cell/organism containing the target gene allows the presentinvention to be used in high throughput screening (HTS). For example,individual clones from the library can be replicated and then isolatedin separate reactions, but preferably the library is maintained inindividual reaction vessels (e.g., a 96 well microtiter plate) tominimize the number of steps required to practice the invention and toallow automation of the process. Solutions containing the product d-siRNAs that are capable of inhibiting the different expressed genes can beplaced into individual wells positioned on a microtiter plate as anordered array, and intact cells/organisms in each well can be assayedfor any changes or modifications in behavior or development due toinhibition of target gene activity.

The d-siRNA can be fed directly to, injected into, the cell/organismcontaining the target gene. The d-siRNA construct may be directlyintroduced into the cell (i.e., intracellularly); or introducedextracellularly into a cavity, interstitial space, into the circulationof an organism, introduced orally, or may be introduced by bathing anorganism in a solution containing the d-siRNA. Methods for oralintroduction include direct mixing of RNA with food of the organism.Physical methods of introducing nucleic, acids include injectiondirectly into the cell or extracellular injection into the organism ofan RNA solution. The d-siRNA may be introduced in an amount which allowsdelivery of at least one copy per cell. Higher doses (e.g., at least 5,10, 100, 500 or 1000 copies per cell) of d-siRNA material may yield moreeffective inhibition; lower doses may also be useful for specificapplications. Inhibition is sequence-specific in that nucleotidesequences corresponding to the duplex region of the RNA are targeted forgenetic inhibition.

The function of the target gene can be assayed from the effects it hason the cell/organism when gene activity is inhibited. This screeningcould be amenable to small subjects that can be processed in largenumber, for example, tissue culture cells derived from invertebrates orinvertebrates, mammals, especially primates, and most preferably humans.

If a characteristic of an organism is determined to be geneticallylinked to a polymorphism through RFLP or QTL analysis, the presentinvention can be used to gain insight regarding whether that geneticpolymorphism might be directly responsible for the characteristic. Forexample, a fragment defining the genetic polymorphism or sequences inthe vicinity of such a genetic polymorphism can be amplified to producea dsRNA from which d-siRNA is prepared according to the subject methods,which d-siRNA can be introduced to the organism or cell, and whether analteration in the characteristic is correlated with inhibition can bedetermined.

The present invention is useful in allowing the inhibition of essentialgenes. Such genes may be required for cell or organism viability at onlyparticular stages of development or cellular compartments. Thefunctional equivalent of conditional mutations may be produced byinhibiting activity of the target gene when or where it is not requiredfor viability. The invention allows addition of d-siRNA at specifictimes of development and locations in the organism without introducingpermanent mutations into the target genome.

In situations where alternative splicing produces a family oftranscripts that are distinguished by usage of characteristic exons, thepresent invention can target inhibition through the appropriate exons tospecifically inhibit or to distinguish among the functions of familymembers. For example, a hormone that contained an alternatively splicedtransmembrane domain may be expressed in both membrane bound andsecreted forms. Instead of isolating a nonsense mutation that terminatestranslation before the transmembrane domain, the functional consequencesof having only secreted hormone can be determined according to theinvention by targeting the exon containing the transmembrane domain andthereby inhibiting expression of membrane-bound hormone.

Therapeutic Applications

The subject d-siRNA compositions produced by the subject methods alsofind use in a variety of therapeutic applications in which it is desiredto selectively modulate, e.g., one or more target genes in a host, e.g.,whole mammal, or portion thereof, e.g., tissue, organ, etc, as well asin cells present therein. In such methods, an effective amount of ad-siRNA composition is administered to the host or target portionthereof. By effective amount is meant a dosage sufficient to selectivelymodulate expression of the target gene(s), as desired. As indicatedabove, in many embodiments of this type of application, the subjectmethods are employed to reduce/inhibit expression of one or more targetgenes in the host or portion thereof in order to achieve a desiredtherapeutic outcome.

Depending on the nature of the condition being treated, the target genemay be a gene derived from the cell, an endogenous gene, apathologically mutated gene, e.g. a cancer causing gene, one or moregenes whose expression causes or is related to heart disease, lungdisease, alzheimer's disease, parkinson's disease, diabetes, arthritis,etc.; a transgene, or a gene of a pathogen which is present in the cellafter infection thereof, e.g., a viral (e.g., HIV-Human ImmunodeficiencyVirus; HBV-Hepatitis B virus; HCV-Hepatitis C virus; Herpes-simplex 1and 2; Varicella Zoster (Chicken pox and Shingles); Rhinovirus (commoncold and flu); any other viral form) or bacterial pathogen. Depending onthe particular target gene and the dose of d-siRNA delivered, theprocedure may provide partial or complete loss of function for thetarget gene. Lower doses of injected material and longer times afteradministration of d-siRNA may result in inhibition in a smaller fractionof cells.

The subject methods find use in the treatment of a variety of differentconditions in which the modulation of target gene expression in amammalian host is desired. By treatment is meant that at least anamelioration of the symptoms associated with the condition afflictingthe host is achieved, where amelioration is used in a broad sense torefer to at least a reduction in the magnitude of a parameter, e.g.symptom, associated with the condition being treated. As such, treatmentalso includes situations where the pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g. preventedfrom happening, or stopped, e.g. terminated, such that the host nolonger suffers from the condition, or at least the symptoms thatcharacterize the condition.

A variety of hosts are treatable according to the subject methods.Generally such hosts are “mammals” or “mammalian,” where these terms areused broadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the hosts will be humans.

The present invention is not limited to modulation of expression of anyspecific type of target gene or nucleotide sequence. Representativeclasses of target genes of interest include but are not limited to:developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors,cytokines/lymphokines and their receptors, growth/differentiationfactors and their receptors, neurotransmitters and their receptors);oncogenes (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB,EBRB2, ETSI, ETS1, ETV6, FOR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN,MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM 1, PML, RET, SRC, TALI,TCL3, and YES); tumor suppressor genes (e.g., APC, BRCA 1, BRCA2, MADH4,MCC, NF 1, NF2, RB 1, TP53, and WTI); and enzymes (e.g., ACC synthasesand oxidases, ACP desaturases and hydroxylases, ADP-glucosepyrophorylases, ATPases, alcohol dehydrogenases, amylases,amyloglucosidases, catalases, cellulases, chalcone synthases,chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNApolymerases, galactosidases, glucanases, glucose oxidases, granule-boundstarch synthases, GTPases, helicases, hemicellulases, integrases,inulinases, invertases, isomerases, kinases, lactases, Upases,lipoxygenases, lyso/ymes, nopaline synthases, octopine synthases,pectinesterases, peroxidases, phosphatases, phospholipases,phosphorylases, phytases, plant growth regulator synthases,polygalacturonases, proteinases and peptidases, pullanases,recombinases, reverse transcriptases, RUBISCOs, topoisomerases, andxylanases); chemokines (e.g. CXCR4, CCR5), the RNA component oftelomerase, vascular endothelial growth factor (VEGF), VEGF receptor,tumor necrosis factors nuclear factor kappa B, transcription factors,cell adhesion molecules, Insulin-like growth factor, transforming growthfactor beta family members, cell surface receptors, RNA binding proteins(e.g. small nucleolar RNAs, RNA transport factors), translation factors,telomerase reverse transcriptase); etc.

As indicated above, the d-siRNA can be introduced into the targetcell(s) using any convenient protocol, where the protocol will varydepending on whether the target cells are in vitro or in vivo.

Where the target cells are in vivo, the d-siRNA can be administered tothe host comprising the cells using any convenient protocol, where theprotocol employed is typically a nucleic acid administration protocol,where a number of different such protocols are known in the art. Thefollowing discussion provides a review of representative nucleic acidadministration protocols that may be employed. The nucleic acids may beintroduced into tissues or host cells by any number of routes, includingmicroinjection, or fusion of vesicles. Jet injection may also be usedfor intra-muscular administration, as described by Furth et al. (1992),Anal Biochem 205:365-368. The nucleic acids may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992), Nature 356:152-154), where gold microprojectiles arecoated with the DNA, then bombarded into skin cells.

For example, the d-siRNA agent can be fed directly to, injected into,the host organism containing the target gene. The agent may be directlyintroduced into the cell (i.e., intracellularly); or introducedextracellularly into a cavity, interstitial space, into the circulationof an organism, introduced orally, etc. Methods for oral introductioninclude direct mixing of RNA with food of the organism. Physical methodsof introducing nucleic acids include injection directly into the cell orextracellular injection into the organism of an RNA solution.

In certain embodiments, a hydrodynamic nucleic acid administrationprotocol is employed. Where the agent is a ribonucleic acid, thehydrodynamic ribonucleic acid administration protocol described indetail below is of particular interest. Where the agent is adeoxyribonucleic acid, the hydrodynamic deoxyribonucleic acidadministration protocols described in Chang et al., J. Virol. (2001)75:3469-3473; Liu et al., Gene Ther. (1999) 6:1258-1266; Wolff et al.,Science (1990) 247: 1465-1468; Zhang et al., Hum. Gene Ther. (1999)10:1735-1737: and Zhang et al., Gene Ther. (1999) 7:1344-1349; are ofinterest.

Additional nucleic acid delivery protocols of interest include, but arenot limited to: those described in U.S. Patents of interest include U.S.Pat. Nos. 5,985,847 and 5,922,687 (the disclosures of which are hereinincorporated by reference); WO/1 1092;. Acsadi et al., New Biol. (1991)3:71-81; Hickman et al., Hum. Gen. Ther. (1994) 5:1477-1483; and Wolffet al., Science (1990) 247: 1465-1468; etc.

Depending n the nature of the d-siRNA, the active agent(s) may beadministered to the host using any convenient means capable of resultingin the desired modulation of target gene expression. Thus, the agent canbe incorporated into a variety of formulations for therapeuticadministration. More particularly, the agents of the present inventioncan be formulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants and aerosols. As such,administration of the agents can be achieved in various ways, includingoral, buccal, rectal, parenteral, intraperitoneal, intradermal,transdermal, intracheal, etc., administration.

In pharmaceutical dosage forms, the agents may be administered alone orin appropriate association, as well as in combination, with otherpharmaceutically active compounds. The following methods and excipientsare merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the nature of the deliveryvehicle, and the like. Preferred dosages for a given compound arereadily determinable by those of skill in the art by a variety of means.

Kits

Also provided are reagents and kits thereof for practicing one or moreof the above-described methods. The subject reagents and kits thereofmay vary greatly. Typically, the kits at least include a Dicerpreparation, as described above. The subject kits may also include oneor more additional reagents, e.g., reagents employed in generating dsRNAas described above, dsRNA, etc.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental I. Materials and Methods A. Dicer Expression andPurification.

The coding region of human Dicer (Acc. No. NM 030621) with N-terminalHis₆- and T7-epitope tags was expressed in Hi5 cells(Invitrogen-BTI-TN-5B1-4) using the Bac-to-Bac expression system(Gibco-pFastbac-HTC). After 48 hrs of expression, cell pellets wereeither stored at −80° C. or lysed in extraction buffer (300 mM NaCl, 50mM NaPO₄, 1% NP-40, pH 8.0) by sonication. Cellular debris and insolubleproteins were removed by centrifugation at 10,000 g. r-Dicer wasaffinity purified in batch from the total soluble protein using Talon™resin (Clontech) as recommended by manufacturer. Briefly, r-Dicer wasbound to Talon™ resin at 4° C. for 1 hr, washed twice (500 mM NaCl, 50mM NaPO₄, 1% NP-40, pH 8.0), washed a third time (500 mM NaCl, 50 mMNaPO₄, pH 8.0), and eluted (500 mM NaCl, 50 mM NaPO₄, 150 mM imidazole,pH 8.0). Further purification of r-Dicer using HiTrap Q HP (Amersham)was done according to manufacturer with a linear elution gradient(Buffer A: 25 mM NaCl, 50 mM NaPO₄, pH 8.0; Buffer B: 1 M NaCl, 50 mMNaPO₄, pH 8.0). Slide-A-Lyzers (Pierce) were used for dialysis asrecommended by manufacturer (500 mM NaCl, 60 mM HEPES, 0.1 mM EDTA, pH8.0). Samples were electrophoresed on 6% (100:1) polyacrylamide gels andwere either Coomassie-stained or transferred to PVDF (Amersham). Themouse α-T7-HRP antibody (Novagen) was used for western blotting asdirected by manufacturer (1:5000).

B. RNA Preparation.

Large dsRNAs (˜500 bp) were made by annealing single stranded RNAs thatwere in vitro transcribed from SP6 and T7 promoter sequences at eitherend of a template generated by PCR. (Alternatively, T7 promotersequences can be employed at both ends to obviate a separate annealingstep). Annealing was carried out by heating sense and antisense RNA (3μM each) at 95° C. for 1 min and then incubating overnight at roomtemperature in 20 mM NaCl, 10 mM HEPES pH 8.0. Chemically synthesizedsiRNAs were synthesized by Dharmacon and were deprotected and providedby Greg Hannon (Cold Spring Harbor Laboratory), as previously described(Elbashir, S. M., et al., Genes Dev. 15,188-200. (2001)). The accessionnumbers and regions targeted are as follows and correspond to the regionrelative to the start codon. For GL3 (Acc. No. U47296), the chemicallysynthesized siRNA targeted 153-173 and the d-siRNAs targeted 113-614.For RL (Acc. No. AF025846) the d-siRNAs targeted the 118-618 region. ForGFP (Acc. No. U55761), the chemically synthesized siRNA targeted 83-103and the d-siRNAs targeted 69-568. For VegT (Acc. No. U59483) thed-siRNAs targeted 733-1377.

C. In Vitro Dicing.

For analysis of r-Dicer activity, typically 500 ng of dsRNA (500 bp) wascombined with lysate or a column fraction that did or did not containr-Dicer (reaction contained: 250 mM NaCl, 30 mM HEPES, 0.05 mM EDTA, 1mM ATP, 2.5 mM MgCl₂, pH 8.0 where half of the volume consisted oflysate or column fraction) and incubated for 12-16 hrs at 37° C. Forsome of the reactions shown in FIG. 1C, these standard conditions weremodified by including 20 U of RNasin (Promega), by leaving outMg²⁺and/or ATP, or by substituting 500 ng of ssRNA or dsDNA for thedsRNA. Reaction was quenched by the addition of 10 mM EDTA, treated withproteinase K (0.2 mg/ml), phenol-chloroform-isoamyl alcohol extracted,and electrophoresed in a 15% polyacrylamide native gel (29:1acrylamide:bisacrylamide).

Gel was cast in 1×TBE, pH 8.5 and electrophoresed in 0.5×TBE pH 8.5 at4° C. For production of d-siRNAs typically 5 μg of dsRNA was incubatedwith r-Dicer (same reaction conditions as above but r-Dicer comprisedonly 10% of reaction volume). Reaction was quenched with 10 mM EDTA,desalted using G-25 spin column (Amersham), deproteinated using EZ-pure(Millipore), and any remaining large dsRNA was removed using Micron-100(Millipore). Electrophoresis in a 15% acrylamide native gel (asdescribed above) was carried out to verify that the d-siRNAs were notcontaminated with large dsRNAs.

To determine the precise size of d-siRNAs (FIG. 2A), ³²P-labeled RNA waselectrophoresed on an 18% (wt/vol) denaturing polyacrylamide sequencinggel. Size standards were generated by alkaline hydrolysis of ³²P-labeledsingle stranded Xenopus β-actin mRNA. For more routine analyses,d-siRNAs were electrophoresed on a 15% (wt/vol) polyacrylamide gel witha synthetic 21 bp siRNA as a size standard.

D. 5′-Phosphorylation of d-siRNAs. A synthetic siRNA (GL3) and variousd-siRNAs (15 pmol) were incubated at 37° for 1 h in shrimp alkalinephosphatase (SAP) buffer (USB) in the presence or absence of 15 U SAP(USB). SAP was inactivated by heating at 65° C. for 15 minutes in thepresence of 10 mM EDTA. The siRNA and d-siRNAs were subsequentlyincubated with 10 U T4 polynucleotide kinase (New England Biolabs) and10 μCi [γ-³²P]ATP (Amersham). Reaction products were analyzed by 15%(wt/vol) native PAGE followed by ethidium bromide staining andphosphorimaging.

E. Cell Culture and Transfections.

The bacmid encoding r-Dicer was generated by recombination in DH10Baccells (Invitrogen). Virus encoding r-Dicer was generated (bytransfection of bacmid with cellFECTIN, Invitrogen) and amplified in Sf9cells (Invitrogen-IPLB-SF-21-AE) as directed by manufacturer. Hi5 cells(Invitrogen-BTI-TN-5B1-4) were grown and infected as instructed bymanufacturer (Invitrogen).

293 and RBL cells were grown at 37° C., in 10% CO₂, and in Dulbecco'smodified Eagle's medium (D-MEM) supplemented with 10% FBS, 100 units/mlpenicillin, and 100 μg/ml streptomycin. 24 hours before transfection,cells were plated onto 24 well plates at 90% confluence in D-MEMsupplemented with 10% FBS, without antibiotics. Co-transfections of 1 μgpGL3-SV40 (Promega), 5 ng pRL-CMV (Promega), and either 210 ng d-siRNAsor 210 ng chemically synthesized siRNAs were carried out in 24 wellplates using Lipofectamine 2000 (Invitrogen) as described by themanufacturer. Cells were lysed 20 hours post transfection and analyzedfor luciferase activity (Dual-luciferase kit, promega).

For silencing endogenous genes, HEK 293 cells were transferred into12-well plates at 50% confluence the day before transfection. d-siRNAswere transfected using Genesilencer reagent (Gene Therapy Systems, SanDiego). Cells were lysed 72 hours post-transfection by vortexing in coldlysis buffer (0.42 M NaCl, 100 mM Tris pH 7.9, 0.5% Triton X-100, 1 mMEDTA, 1 mM EGTA) supplemented with protease inhibitors (aprotonin 1μg/ml, 0.2 mM PMSF, pepstatin, leupeptin, and chymostatin all at 0.2μg/ml). Lysates (˜60 μg protein) were subjected to SDS-PAGE andtransferred to PVDF (Amersham). Blots were probed with variousantibodies: actin (1:200, Santa Cruz), Cdc25A (1:500, Santa Cruz),Cdc25C (1:500, Santa Cruz), cyclin E1 (1:500, Santa Cruz) orphospho-elF2α (1:500, Biosource International). Secondary antibodieswere conjugated to either alkaline phosphatase (AP; Sigma) orhorseradish peroxidase (HRP; Amersham) and used at 1:2500. TheImmun-Star HRP chemiluminescent detection kit (Bio-Rad) was used for HRPdetection and CDP-Star (Tropix-Perkin Elmer) for AP.

F. Activity Assay

293 cells were transfected with a plasmid encoding Dicer with twoN-terminal tags, His₆ and the T7 epitope or an irrelevant plasmid as anegative control. After 3 days of expression cells were lysed inimmunoprecipitation buffer. Dicer was immunoprecipitated with a T7antibody. The agarose-antibody conjugate was then incubated with 40 pmolof internally labeled 500 bp dsRNA for 2.5 hr in normal reactionconditions. Various amounts of r-Dicer (1, 2.5, 5, 10, 15 μl) were alsoincubated with 40 pmol of internally labeled 500 bp dsRNA under the sameconditions. The RNA was then electrophoresed in a 15% nativepolyacrylamide gel. The amount of d-siRNA produced was then determinedby phosphorimaging. To determine the specific activity the amount ofprotein that cleaves a percentage of dsRNA is measured. Protein amountis measured by western blotting.

II. Results

Full-length, His₆- and T7-tagged-Dicer was expressed in Hi5 insect cellsby baculovirus infection and purified on a cobalt Sepharose column. Themain protein eluted was a 225 kDa protein, which corresponds to thepredictedmolecular mass for Dicer (FIG. 1A, top panel, left side). The225 kDa band co-migrated with the main immunoreactive band on ananti-tag immunoblot, confirming its identity as recombinant Dicer (FIG.1A, middle panel, left side).

It was then determined that the purified recombinant Dicer was able toprocess dsRNA into siRNAs. We incubated samples with a 500 bp dsRNA,submitted the reaction products to native polyacrylamide gelelectrophoresis, and looked for the production of ˜22 bp products. Thepurified Dicer efficiently converted the dsRNA to 20-21-22 bp forms(FIG. 1A, bottom panel, left side; FIG. 2A).

Because Drosophila extracts are capable of generating siRNAs, it islikely that the Hi5 insect cells used to produce r-Dicer contained someendogenous Dicer activity. Therefore, we addressed the question ofwhether the Dicer activity in our purified fractions was due to ther-Dicer or to some co-purifying insect protein. We subjected lysatesfrom uninfected Hi5 cells to the same purification procedure andassessed fractions for Dicer activity. A small amount of RNA processingactivity could be detected in the total soluble Hi5 cell lysate and inthe flow through from the cobalt affinity column (FIG. 1 A, bottompanel, right side). However this activity was not detected in the columneluates, indicating that it could not account for the high levels ofDicer activity seen in the purified r-Dicer preparation. These dataindicate that the r-Dicer is responsible for the Dicer activity seen inthe purified fractions.

Previous in vitro data demonstrated that Dicer is capable of interactingwith other proteins implicated in RNA interference. Thus, it seemedplausible that active r-Dicer might be a complex consisting of r-Dicerand some endogenous insect cell proteins. Since the r-Dicer is highlyoverexpressed in the Hi5 cells, this putative complex would likelyaccount for a small proportion of the total expressed r-Dicer, and sothe bulk of the r-Dicer protein might be chromatographically separablefrom the active r-Dicer. We therefore subjected the purified, activer-Dicer to Q-Sepharose ion exchange chromatography and assessed whetheror not the r-Dicer protein and Dicer activity co-eluted. As shown inFIG. 1B, the protein and activity did co-elute. Thus, there is noevidence that r-Dicer requires other proteins for activity.

r-Dicer was subjected to various reaction conditions to determinecofactor and substrate specificity as well as to ensure that the 20-21-mers were generated by r-Dicer and not a nonspecific RNase or nuclease.dsRNA was efficiently processed by r-Dicer (FIG. 1C, lane 1) but dsDNAwas not (FIG. 1C, lane 2), establishing that r-Dicer is RNA-specific.r-Dicer generated small amounts of 22 bp product from ssRNA, which maybe due to cleavage of hairpins or contaminating dsRNA (FIG. 1C, lane 3,and data not shown). r-Dicer was not inhibited by RNasin (FIG. 1C, lane4), distinguishing it from most ribonucleases. Mg²⁺ was required forr-Dicer activity (FIG. 1C, lanes 6 and 7), but ATP was not (FIG. 1C,lane 5). In contrast, other workers have found that ATP is important forRNAi and dicing in extracts and in Dicer immunoprecipitates (Zamore, etal., Cell 101, 25-33. (2000); Bernstein, et al., Nature 409, 363-366.(2001).; Nykanen, et al., Cell 107, 309-321. (2001)). The reason forthis difference is unclear, but it is possible that ATP is moreimportant for the activity of crude Dicer preparations than it is forpurified Dicer preparations. High concentrations of manganese (2.5-5 mM)and calcium (10 mM) inhibited r-Dicer activity (data not shown).

siRNAs produced in Drosophila extracts are 5′-phosphorylated, and thisis important for gene silencing (Elbashir, S. M., Lendeckel, W. &Tushcl, T. RNA interference is mediated by 20-, 21- and 22-nucleotideRNAs. Genes Dev 15, 188-200. (2001)). To determine if d-siRNAs are5′-phosphorylated, d-siRNAs were incubated with or without shrimpalkaline phosphatase (SAP), followed by T4 polynucleotide kinase and[γ-³²P]ATP. SAP-dephosphorylated d-siRNAs were phosphorylated by T4polynucleotide kinase, but mock-dephosphorylated d-siRNAs were not (FIG.2B), indicating that d-siRNAs are quantitatively monophosphorylated attheir 5′-ends. A synthetic siRNA, which has a 5′-hydroxyl group ratherthan a 5′-phosphate, was phosphorylated by T4 polynucleotide kinase withor without SAP treatment (FIG. 2B).

To explore the mechanism of in vitro dicing and determine whether usefulamounts of d-siRNAs could be produced, we quantified d-siRNA productionas a function of time and Dicer concentration (FIG. 2C-H). Substantialyields were obtained; at the highest Dicer concentration used (75 nM),˜70% of the starting material was converted to d-siRNAs within 24 h(FIG. 2C, H). The amount of product produced (50 pmol) greatly exceededthe amount of Dicer used (0.75 pmol) (FIG. 2C, H), indicating thatprocessing was enzymatic rather than stoichiometric. The high yieldsalso imply that r-Dicer must produce a complex pool of 20-21 bp productsrather than one or two predominant products. dsRNAs intermediate betweenthe 500 bp substrate and 20-21 bp product were not detected (FIG. 2C),suggesting that r-Dicer cleaves dsRNA processively.

We next assessed whether d-siRNAs were capable of specific genesilencing. d-siRNAs and siRNAs (FIG. 3A) were co-transfected withfirefly and Renilla luciferase constructs into HEK 293 cells, and after20 h of expression, firefly (FIG. 3B) and Renilla (FIG. 3C) luciferaseactivities were assayed individually (Elbashir, S. M. et al. Duplexes of21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature 411, 494-498. (2001)). Diced GL3 siRNAs were found to becomparable to a highly effective synthetic siRNA in inhibiting fireflyluciferase expression, and control siRNAs (a synthetic GFP siRNA andVegT and RL d-siRNAs) had no effect (FIG. 3B). RL d-siRNAs inhibitedRenilla luciferase expression, and again the control GFP siRNA and VegTd-siRNAs had no effect (FIG. 3C). GL3 d-siRNAs caused some inhibition ofRenilla luciferase expression (FIG. 3C; see also FIG. 3F for a secondset of experiments). This may represent an off-target effect; however,as noted above, RL d-siRNAs had no effect on firefly luciferaseexpression, so off-target effects are not an inevitable consequence ofusing d-siRNAs (also note that VegT (FIG. 3B,C) and GFP (FIG. 3E,F)d-siRNAs do not affect either form of luciferase at lowerconcentrations). Both the synthetic and diced GL3 siRNAs shifted theratio of firefly-to-Renilla luciferase expression toward Renillaluciferase, with the synthetic GL3 siRNA being somewhat more selectivethan the diced GL siRNA, and the RL d-siRNAs shifted the ratio towardsfirefly luciferase (FIG. 3D). Thus, both of the d-siRNAs were selectivefor the correct species.

Concentrations of synthetic siRNAs as low as 1.5 nM are sometimescapable of specific gene silencing (Elbashir, S. M. et al. Duplexes of21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature 411, 494-498. (2001)). However, many synthetic siRNAs are lesseffective, and so it was of interest to see how d-siRNA pools comparedto a highly effective siRNA in potency. To ensure silencing observedwith d-siRNAs was not due to contaminating large dsRNA left over afterM-100 chromotography, d-siRNAs were also gel purified, and the twodistinct preps were compared. As shown in FIG. 3E (dark gray bars), thesynthetic and diced GL3 siRNAs all decreased firefly luciferaseexpression by about 90% at the lowest concentration (3 nM) tested, andshowed greater silencing at higher concentrations. Thus, the potency ofthe GL3 d-siRNA pools compared well to that of the synthetic GL3 siRNA.Gel-purified and M-100-purified d-siRNAs were similar to each other inefficacy, suggesting that effective gene silencing is not affected bypurification strategy (FIG. 3E).

We compared the non-specific effects of diced and synthetic siRNAs byexamining the effect of GFP siRNAs on the expression of firefly andRenilla luciferases. Neither the synthetic nor the diced GFP siRNAs (gelpurified or M-100 purified) significantly inhibited GL3 luciferase (FIG.3E) or Renilla luciferase (FIG. 3F) expression at concentrations below30 nM. At higher concentrations, all three GFP siRNA preparationsnon-specifically silenced both luciferases, with the synthetic GFP siRNAcausing the least silencing and the gel-purified GFP d-siRNA the most(FIG. 3E, F). Thus, the siRNA and d-siRNAs had similar concentrationwindows for specific gene silencing, and none was free of non-specificeffects at concentrations of 100 nM and greater. Some of thenon-specific silencing was probably due to an interferon response, asindicated by elevated levels of elF2α phosphorylation (Stark, G. R.,Kerr, I. M., Williams, B. R., Silverman, R. H. & Schreiber, R. D. Howcells respond to interferons. Annu Rev Biochem 67, 227-264. (1998))(e.g. FIG. 3H, the 100 and 300 nM M-100 d-GFP samples). However, somesamples showed little elevation of elF2α phosphorylation, but stillshowed a non-specific decrease in luciferase expression (e.g. FIG. 3H,the 100 and 300 nM gel-purified d-GFP samples), indicating thatmechanisms other than the interferon response contributed to thedecreased luciferase expression. Gel-purified and M-100-purifiedd-siRNAs were generally similar in toxicity (FIG. 3E-G), althoughincreases in elF2α phosphorylation were seen with some of theM-100-purified d-siRNAs, but not with gel purified d-siRNAs (FIG. 3H).The diced and synthetic GL3 siRNAs all showed selectivity for fireflyluciferase over Renilla luciferase at both low and high siRNAconcentrations (FIG. 3G, dark gray bars).

Next we assessed whether d-siRNAs can inhibit the expression ofendogenous genes. The first target examined was cyclin E1, theregulatory subunit of the Cdk2/cyclin E1 complex. HEK 293 cells weretransfected with two concentrations of cyclin E1 d-siRNAs, an irrelevantcontrol d-siRNA (RL luciferase), or water, and the amount of endogenouscyclin E1 protein remaining after 72 hours was assessed. As shown inFIG. 4A, 30 nM d-siRNAs caused some decrease in cyclin E1 levels, and 60nM d-siRNA largely eliminated the cyclin E1 protein. Little elF2αphosphorylation was seen in cells treated with cyclin E1 d-siRNAs (FIG.4A). A higher level of elF2α phosphorylation was seen in cells treatedwith RL d-siRNAs (FIG. 4A), probably due to the persistence of the RLd-siRNAs in cells that do not possess an RL luciferase target.Nevertheless, even this extent of elF2α phosphorylation did not resultin a detectable decrease in cyclin E1 protein levels (FIG. 4A),indicating that an interferon response could not account for thedecreased cyclin E1 levels seen in cyclin E1 d-siRNA-treated cells.

As a second target we examined Cdc25C. Cdc25C is one of threeclosely-related phosphatases (Cdc25A, B, and C) that dephosphorylate andactivate cyclin-dependent kinases, allowing us to address the questionof off-target gene silencing again in a more natural context. dsRNAscorresponding to the entire coding regions of Cdc25C were diced invitro. The d-siRNAs were gel purified and transfected into HEK 293cells, and the expression of Cdc25C and Cdc25A was assessed byimmunoblotting. The Cdc25C d-siRNAs decreased Cdc25C by about 90%,without increasing elF2α phosphorylation (FIG. 4B). Expression of Cdc25Awas unaffected (FIG. 4B), even though Cdc25C and Cdc25A share stretchesof 13-15 consecutive identical nucleotides and several longer stretcheswith single mismatches. d-siRNAs derived from an irrelevant B-Raf cDNAhad no effect on either Cdc25C or Cdc25A expression (FIG. 4B). ThusCdc25C d-siRNAs specifically silenced the expression of Cdc25C.

In summary, r-Dicer processes large dsRNAs into 20-21 bp siRNAs suitablefor gene silencing studies in mammalian cells. Yields are high, up to70%, and the products of the Dicer reaction are easily purified awayfrom any residual large dsRNA by M-100 chromatography or gelpurification. Diced siRNAs are comparable in potency to syntheticsiRNAs, and are similar in terms of non-specific toxicity as well. Forsome purposes (e.g. knockdowns of splice variants, stable knockdowns, orconditional knockdowns), individual siRNAs may still be preferable tod-siRNAs. However, for general functional studies, d-siRNAs are simpleto produce, effective in gene silencing, and easily scalable, makingthem a useful addition to the siRNA arsenal.

III. Representative In Vitro Dicing-Single Gene Silencing Protocol

The following representative protocol describes a way to specificallysilence a single gene in cell culture according to the subjectinvention. The following protocol is based on the previous finding thatsmall interfering RNAs (siRNAs) induce gene silencing in cultured cells(Elbashir, et al., supra) and an RNase III family enzyme, Dicer, iscapable of cleaving larger dsRNAs into 22-mers (Bernstein, et al.,Nature 409, 363-366. (2001)). Purified recombinant Dicer (r-Dicer) isused to cleave larger dsRNAs into d-siRNAs. The general strategy is topick a region in Your Favorite Gene (YFG), make a template forgenerating large dsRNA in vitro, incubate dsRNA with r-Dicer, purifyd-siRNAs, and introduce into cells.

A. r-Dicer Purification

r-Dicer is produced by infection of Hi5 cells with baculovirus and issubsequently purified, in batch, using cobalt resin.

1. Infect Hi5 cells with 100 μl of virus per 1×10⁷ cells and express for46-48 hours. Pellets can be directly subjected to purification or storedat −80° C. Typically 1×10⁸ cells are infected with 1 ml of virus.Protein production is efficient in either adherent or suspensioncultures.2. Pellets (1×10⁸ cells) are resuspended in 10 ml of α-extractionbuffer. Add protease inhibitors to a final concentration of: 1 mM PMSF:0.55 μg/ml leupeptin; 0.55 μg/ml pepstatin; 1 μg/ml aprotonin

-   -   α-pH 8.0    -   50 mM NaPO₄    -   300 mM NaCl    -   1% NP-40        3. Cells are lysed by sonication, 5×10 second pulses at 40% duty        cycle, in an ice slurry. Allowing samples to chill for 30        seconds between the 10-second pulses prevents heating of the        sample.        4. Cell debris and insoluble proteins are removed by        centrifugation at 10,000×g for 20 minutes at 4° C. About 50% of        r-Dicer is present in the soluble fraction        5. The 10,000×g supernatant is mixed (using a conventional        rotator) with a 500 μl bed volume of cobalt Sepharose (Talon,        Clontech; capacity 3 mg/1 ml bed volume) for 1 hour at 4° C. to        allow binding of r-Dicer to the resin. Note: the resin is washed        twice with 20 volumes of α-extraction buffer; resin is collected        by centrifugation at 700×g.        6. After binding, the resin is again collected by centrifugation        at 700×g and 4° C. for 5 minutes.        8. Resin is washed twice by mixing for 10 minutes at 4° C. with        20 volumes of β-wash buffer. Resin is collected by        centrifugation at 700×g and 4° C. for 2 minutes.    -   β-pH 8.0    -   50 mM NaPO₄    -   500 mM NaCl    -   1% NP-40        8. 2 additional washes with β-minus buffer are used to remove        the detergent (NP-40). Washes are performed as in step 7.    -   β-pH 8.0    -   50 mM NaPO₄    -   500 mM NaCl        9. r-Dicer is eluted by five successive elutions with 500 μl of        γ-elution buffer. Between elutions resin is collected by        centrifugation at 700×g and 4° C. for 2 minutes.    -   γ-pH 8.0    -   50 mM NaPO₄    -   500 mM NaCl    -   150 mM imidazole        10. The elution fractions are dialyzed (Slide-A-lyzer, Pierce)        at 4° C. against 3 L of dialysis buffer. Three successive 1 L        dialyses are performed, the first two for two hours and the        third overnight. r-Dicer is stored at 4° C.    -   Dialysis-pH 8.0    -   60 mM Hepes pH 8.0    -   500 mM NaCl    -   0.1 mM imidazole        11. The 225 kDa r-Dicer can be detected by Coomassie-staining or        Western blotting (anti-T7 or anti-His) after electrophoresis in        a 6%, 100:1 SDS-polyacrylamide gel.

To obtain higher protein yields, the above protocol can be modified tostart with more material, e.g., 4×10⁸ cells.

B. Activity Assay

1. r-Dicer activity can be assayed by setting up a Dicer reaction. About80% of the large dsRNA will be cleaved into d-siRNAs when the followingreaction conditions are used.

-   -   Reaction Conditions    -   X μl dsRNA (500 ng)    -   1.0 μl 10 mM ATP*    -   0.5 μl 50 mM MgCl₂    -   4.0 μl Rxn Dil. Bfr.*    -   1.0 μl Enzyme    -   X μl H₂O    -   10.0 μl    -   *pH 8.0; Incubate @ 37° C. 18-22 hours        2. After overnight incubation the reaction is quenched with EDTA        to a final concentration of 10 mM and extracted with        phenol-chloroform-isoamyl alcohol (25:24:1). The RNA is then        electrophoresed in a 15% native polyacrylamide gel (29:1, cast        in 1× and electrophoresed in 0.5×TBE) at 10 Watts at 4° C. RNA        is visualized by staining with ethidium after electrophoresis.        C. Generation of dsRNA to be Processed by Dicer:        1. To silence YFG the sequence or partial sequence must be        known. A region can be selected that abides to the following        rules.        a. Determining a Region to Target

In one embodiment, a region about 100-150 nucleotides downstream of theATG is selected, which prevents the d-siRNAs from having to compete withtranslation initiation proteins that bind mRNA. However, GreenFluorescent Protein (GFP) was silenced using the entire coding region asa target. Alternatively a region in the 3′ UTR can be used and may havesome advantages. In either case, usually a single gene is to be silencedso there is a worry about silencing several isoforms or silencing genesthat are very similar. Therefore, the region should be lined up withisoforms and variants to make sure the silencing will be specific.

b. Determining the Size of the Target

r-Dicer will process 500 and 1000 bp dsRNAs into d-siRNAs. It is likelythat Dicer will also process much larger dsRNAs, even a full-lengthcDNA. However, for silencing a single gene, a 500 bp dsRNA issufficient.

2. To generate d-siRNAs, a large dsRNA corresponding to the targetregion is generated by in vitro transcription, thus a DNA template ismade. This step can be done several ways, e.g., by making a PCR templatewith T7 promoters at both ends. Alternatively, the region of YFG can besubcloned into a transcription vector with in vitro promoters at bothends and digesting the plasmid after each promoter can generatetemplate. The dsRNA can be made by annealing the single stranded(ssRNAs) or can be made by adding both enzymes to the in vitro reaction(e.g. add SP6 and T7 enzyme to single reaction). This method is moretime consuming and possess no advantages unless YFG is already in avector with promoters at each end and can easily be linearized toproduce a template for the sense strand RNA (sRNA) and the antisensestrand RNA (asRNA). dsRNA can be generated in a variety of other waysbut the most efficient seems to be with PCR template with T7 promoterends. Even if a full length cDNA of YFG is not available, the templatecan be generated from cDNA or a library.a. Designing the Primers

The primers used to produce a template contain a phage promoter sequence(T7, T3, or SP6) followed by gene specific sequence. T7 seems to producethe largest yield but the others also work.

-   -   5′-Primer and 3′-Primer

SEQ ID NOS: 1-3 +1 5′ GCG TAATACGACTCACTATA GG 18-21 gene specificnucleotides 3′    Leader T7 promoter sequence +1 5′ GCGAATTAACCCTCACTAAA GG 18-21 gene specific nucleotides 3′    Leader T3promoter sequence +1 5′ GCG ATTTAGGTGACACTATA GA 18-21 gene specificnucleotides 3′    Leader SP6 promoter sequenceb. Primer Example

These are the primers were used to generate a template for cdc25c(accession number NM001790). cdc25c has two splice variants and variant2 (accession number NM022809) is smaller because it lacks two regions.This template will determine if splice variants can be silenced even ifonly a portion of the d-siRNAs is targeting one variant.

-   -   5′-Primer and 3′-Primer

SEQ ID NOs: 4 to 5 +1 5′ GCG TAATACGACTCACTATA GG AGAGAGACACTTCCTTTACCG3′    Leader T7 promoter sequence 99 bp from ATG +1 5′ GCGTAATACGACTCACTATA GG TATAGGCCACTTCTGCTCACC 3′    Leader T7 promotersequence 634 bp from ATG3. dsRNA is made with MegaScript (Ambion or equivalent from anothercompany). The reaction is completed as recommended by manufacturer(include Dnase I treatment). For most of the dsRNAs tested, annealingoccurs in the transcription reaction and can be monitored byelectrophoresing the dsRNA in a 2% agarose gel. dsRNA will migrate likeDNA, i.e., a 500 bp dsRNA will migrate at the same rate as a 500 bp bandin a DNA ladder, whereas ssRNA will migrate much faster than a dsDNA ofthe equivalent size. It is possible that regions of some genes will notform dsRNAs during the transcription reaction. If this is the case RNAis then annealed directly after the transcription reaction by addingNaCl to 20 mM and Hepes to 10 mM, heating at 95° C. for 1 min 30 sec,and then incubating at 37° C. for 1 hr. It is possible that strandscission may occur when heated in the presence of divalent ions such asMg²⁺. Therefore, EDTA would need to be added before heating. This is aproblem because r-Dicer requires Mg²⁺. Noticeable strand scission hasnot been detected and therefore, RNA is heated as described withoutadding EDTA. Alternatively RNA can be purified as described below priorto annealing, but annealing may not be as efficient (some regions mayrequire divalent cations to anneal properly). Making RNA withFlourescein labeled-UTP, Dicing the dsRNA, and visualizing the cellsafter transfection can determine transfection efficiency.D. Generation of d-siRNAs1. Once dsRNA is made, in vitro dicing can be done. r-Dicer cleavesdsRNA slightly better if the dsRNA is purified. dsRNA can be purified bysubsequent precipitations, first with LiCl and then with NH₄OAC (asdirected by Ambion). Alternatively if a large number of differentd-siRNAs are desired, for example in a high-throughput screen, thepurification step can be omitted because r-Dicer will cleave unpurifieddsRNA, but cleavage is slightly less efficient as compared to purifieddsRNA.

2. Reaction Conditions

-   -   X μl dsRNA (5 μg)    -   9.0 μl 10 mM ATP*    -   4.5 μl 50 mM MgCl₂    -   35.0 μl Rxn Dil. Bfr.*    -   10.0 μl Enzyme    -   X μl H₂O    -   90.0 μl    -   *pH 8.0; Incubate @ 37° C. 18-22 hours    -   Note that the above volume can be scaled up as desired.        3. Several methods can be used to obtain pure d-siRNAs. r-Dicer,        salts, buffer and leftover large dsRNA are removed.        a. “Cookbook” clean up—Using a series of spin columns large        dsRNAs and r-Dicer are completely removed and most of the salts,        NTPs and buffers are removed from the Dicer reaction. This        produces d-siRNAs ready for transfection (in a minimal amount of        salt, buffer, and NTP).        i. Desalt on G-25 spin column (Amersham, or equivalent from        another company) after adding 10 μl of 100 mM EDTA to the 90 μl        reaction.        Manufacturers protocol followed (about 95% efficient).        ii. Extract protein using EZ-pure (Millipore). Manufacturers        protocol followed-Spin 100 μl through, add 10 μl H₂O and spin        again.        iii. Remove large dsRNAs with Microcon M-100 (Millipore) as        directed by manufacturer.        Note that the above protocol can be modified to replace        steps (i) and (ii) with a precipitation step.        b. “Traditional” clean up—Using traditional methods recommended        by Maniatis for nucleic acid purification and concentration        r-Dicer, salts, NTPs and buffers can be almost completely        removed. This produces a d-siRNAs ready for transfection.        i. Extract protein with phenol-chloroform-isoamyl alcohol        (25:24:1) after addition of 10 μl of 100 mM EDTA to the 90 μl        Dicer reaction. Proteinase K treatment is optional, but        typically is unnecessary since only a small amount of protein is        used in the reaction.        ii. Remove large dsRNAs with Microcon M-100 (Millipore) as        directed by manufacturer.        iii. Precipitate by adding MgCl₂ to a final concentration of 10        mM, NH₄OAC to a final concentration of 0.75 M (Maniatis        recommends 2-2.5 M, but this amount does not seem to be        necessary), and 1 volume of Isopropanol (1 volume after the        addition of salt). Vortex and spin a 14,000 RPM at 4° C.        Chilling the solution does not seem to be necessary. Wash with        70% ethanol, dry and resuspend in nuclease free H₂O. It has not        determined whether it is better to precipitate before or after        the removal of large dsRNA.        c. Ambion spin columns—Using a series of spin columns d-siRNAs        can be separated from r-Dicer, salts, buffer, and NTPs. This        produces a d-siRNAs ready for transfection.        i. Add 10 μl of 100 mM EDTA to the 90 μl reaction and purify        using the Ambion column as suggested by manufacturer. This step        will remove r-Dicer, salts, buffer, and NTPs        ii. Remove large dsRNAs with Microcon M-100 (Millipore) as        directed by manufacturer.        iii. Note: it is possible that reversing the order of spin        columns may result in a higher purity and yield of d-siRNAs,        however this has not been tested.        d. After d-siRNAs are purified the concentration can be        determined by measuring the absorbance at 260 nm. Typically 2.5        μg of d-siRNAs are obtained from 5 μg of dsRNA. The        concentration of the d-siRNAs will depend on which method was        used for purification. If the “cookbook” method is used, the        concentration is usually 25 ng/μl. A higher concentration may be        obtained by using a speed vac to decrease the volume or by using        the “traditional” method and resuspending the d-siRNA in a small        volume of H₂O. Note: when using the “cookbook” method there is        residual ATP that absorbs at 260 nm, therefore an appropriate        blank must be used. The blank is a reaction without RNA that has        been purified using the “cookbook” method. In addition, a 15%        native polyacrylamide gel (29:1, cast in 1× and electrophoresed        in 0.5×TBE) should be ran as described above to ensure that the        d-siRNAs are free of larger dsRNAs.        E. Transfect and assay for ablation of protein and appearance of        a phenotype.

IV. Conclusion

The above demonstrates that recombinant Dicer can cleave large dsRNAs(.>500 base pairs) into a random pool of siRNAs (d-siRNAs)in vitro .TheDicer-generated d-siRNAs are capable of gene silencing in mammalian celllines and are at least as effective as chemically synthesized siRNAs,and in some cases more effective. For commonly used mammalian celllines, generating and using d-siRNAs is a more efficient andcost-effective method of gene silencing than the currently availabletechniques using single siRNAs (chemical synthesis, in vitrotranscription, in vivo encoded hairpins)and introduces a new technologythat will allow large-scale functional genomic screening. RNAi hasbecome the method of choice for loss-of-function (lof) investigations innumerous systems including, C.elegans, Drosophila, fungi, plants, andeven mammalian cell lines.

For loss-of-function investigations in mammalian cell lines, usingd-siRNAs instead of single siRNAs has several advantages that areattributed to the fact that a pool of siRNAs is being used instead of asingle siRNA. As previously mentioned using d-siRNAs, instead of singlesiRNAs, is cost-efficient and obviates the need to guess which 21-22nucleotide sequence to target. In addition, single siRNAs would causecleavage of the mRNA at a single site, possibly leaving the 3′-fragmentto be translated as seen with antisense oligodoxynucleotides. TheseN-terminal truncated proteins may act as dominant negative orconstitutively active truncation, potentially altering the trueknock-out phenotype, whereas d-siRNAs will cause cleavage of the 500nucleotide region targeted thereby decreasing the chances of translatinga truncated fragment.

The method we describe here is simple and time-efficient such thatwithin one week, a phenotype for “your favorite protein” may bedetermined. Recombinant Dicer may also allow pools or libraries ofd-siRNAs to be generated and screened. This technology will allowlarge-scale functional genomic investigations in mammalian cell lines.

It is evident from the above results and discussion that the subjectinvention provides improved methods of producing siRNAs, as well asimproved methods of using the produced siRNAs in various applications,including high throughput loss of function applications. As such, thesubject invention makes the rapid determination of gene functionpossible. Accordingly, the present invention represents a significantcontribution to the art.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. The citation of any publication is for its disclosure priorto the filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A method for producing siRNA from an initial dsRNA, said methodcomprising: contacting said dsRNA with a composition enriched for aprotein having an activity that cleaves a dsRNA substrate into fragmentshaving siRNA activity to produce said siRNA, wherein said compositionefficiently produces siRNAs from dsRNA.
 2. The method according to claim1, wherein said method is in vitro.
 3. The method according to claim 1,wherein said contacting occurs in the absence of ATP.
 4. The methodaccording to claim 1, wherein said protein is a recombinantly producedprotein.
 5. The method according to claim 1, wherein said protein isfull-length Dicer or an active fragment thereof.
 6. The method accordingto claim 1, wherein said composition converts at least 80% of said dsRNAto siRNA.
 7. The method according to claim 1, wherein siRNAs produced bysaid method range in length from about 20 to 23 residues.
 8. The methodaccording to claim 7, wherein siRNAs produced by said method are 20-21residues in length.
 9. The method according to claim 1, wherein saidmethod produces a d-siRNA preparation of siRNAs from said dsRNA.
 10. Amethod of at least reducing the expression of a gene in a target cell,said method comprising: producing an siRNA preparation for said geneusing the method of claim 1; and introducing into said cell an effectiveamount of said siRNA preparation to at least reduce expression of saidgene.
 11. The method according to claim 10, wherein said siRNApreparation is a d-siRNA preparation according to claim
 9. 12. Themethod according to claim 10, wherein said method is an in vitro method.13. The method according to claim 10, wherein said method is an in vivomethod.
 14. The method according to claim 10, wherein said method is amethod of silencing expression of said gene.
 15. The method according toclaim 10, wherein said method is a loss of function assay.
 16. A kit foruse in preparing siRNA from dsRNA, said kit comprising: a compositionenriched for a protein having an activity that cleaves a dsRNA substrateinto fragments having siRNA activity to produce said siRNA, wherein saidcomposition efficiently produces siRNAs from dsRNA.
 17. A kit accordingto claim 16, wherein said composition is ATP free.
 18. The kit accordingto claim 16, wherein said protein is a recombinantly produced protein.19. The kit according to claim 16, wherein said protein is full lengthdicer or an active fragment thereof.
 20. The kit according to claim 16,wherein said kit further comprises instructions for practicing a methodaccording to claim
 1. 21. A d-siRNA preparation producing according tothe method claim 1.